Role of the Subtilisin-like Serine Protease CJPRB from Cordyceps javanica in Eliciting an Immune Response in Hyphantria cunea

Hyphantria cunea is a globally distributed quarantine plant pest. In a previous study, the Cordyceps javanica strain BE01 with a strong pathogenic effect on H. cunea was identified, and overexpression of the subtilisin-like serine protease CJPRB of this strain was found to accelerate the death of H. cunea (previous research results). In this study, the active recombinant CJPRB protein was obtained through the Pichia pastoris expression system. It was found that CJPRB protein administration to H. cunea via infectation, feeding and injection was able to induce changes in protective enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and polyphenol oxidase (PPO), and the expression of immune defense-related genes in H. cunea. In particular, CJPRB protein injection induced a more rapid, widespread and intense immune response in H. cunea compared to the other two treatment methods. The results suggest that the CJPRB protein may play a role in eliciting a host immune response during infectation by C. javanica.


Introduction
Entomopathogenic fungi, as natural regulators of pest populations, have an advantage over most entomopathogens in that they can penetrate the insect body surface to infect insects without ingestion [1]. Cordyceps species are a group of globally distributed entomopathogenic fungi that are capable of infecting more than 40 species of insects in 8 orders, exhibiting a broad spectrum of insecticidal activity [2][3][4][5][6][7][8]. Among Cordyceps spp., Cordyceps javanica is capable of infecting insects of 12 genera in 3 orders [9]. The mortality rate of Hyphantria cunea larvae infected by the strain C. javanica BE01 isolated by us previously was over 85% at 1 × 10 8 conidia/mL, indicating that this strain has good application prospects [10].
Typically, disruption of the insect cuticle enables successful infectation by entomopathogenic fungi. This process of cuticle destruction involves the following steps: conidial adhesion, germ tube formation, appressorium formation and penetration peg formation [11,12]. The appressoria release an array of hydrolytic enzymes for cuticle penetration and generate mechanical pressure that allows the fungus to pierce the insect cuticle through the penetration peg [13,14]. After the entomopathogenic fungus penetrates the cuticle and reaches the hemocoel, it sequesters insect nutrients, produces toxins and destroys the host cells, eventually killing the host [15]. Cuticle-degrading enzymes are important factors affecting the penetration ability and virulence of entomopathogenic fungi and include a series of hydrolytic enzymes, such as lipase, chitinase and protease [16,17]. Among them, the subtilisin-like serine protease Pr1 and trypsin-like serine protease Pr2 are the proteases most closely involved in the ability of entomopathogenic fungi to penetrate the cuticle [18]. Pr1 has been shown to be the main and most effective protease involved in

Effects of CJPRB Protein Feeding on the Larvae of H. cunea
After the CJPRB protein was fed to the second-instar larvae of H. cunea for 24 h, the enzyme activity of CAT in H. cunea was found to be significantly higher than that in the control GFP treatment group, increasing by 19.79% (t = 2.887, df = 8, p < 0.05) ( Figure 4A). POD activity in H. cunea fed CJPRB for 36 h and 60 h was not the same as that in the control GFP treatment group, and the difference was significant, with enzyme activities increas-

Discussion
In this study, we successfully expressed an active recombinant protein, CJPRB, through the P. pastoris expression system, and the protein was able to induce an immune defense response in larvae of H. cunea by infectation, feeding and injection.
The P. pastoris expression system is currently the most successful eukaryotic expression system, capable of post-translational processing of the expressed proteins and enabling better protein activity than that obtained via prokaryotic expression [33]. To date, more than 500 exogenous proteins have been successfully expressed in this eukaryotic expression system [34,35]. The activity of the Pr1A protein expressed by Zhang et al. through P. pastoris was 10.6-fold higher than that of the Pr1A protein expressed in a prokaryotic expression system by Wang et al. [34,36]. In the present study, active CJPRB protein was obtained by expression in P. pastoris and could be used in subsequent experiments.
The PPO system plays a key role in insect immune defense by recognizing foreign invading substances. In addition, the protective enzymes SOD, POD and CAT are present in a dynamic balance in insects, and this dynamic balance plays an important role in maintaining the metabolic balance in insects. However, when insects are under stress, the protective enzyme system in the body changes or even disrupts the normal dynamic balance, leading to metabolic disorders in insects. In CJPRB-treated larvae of H. cunea, POD was

Discussion
In this study, we successfully expressed an active recombinant protein, CJPRB, through the P. pastoris expression system, and the protein was able to induce an immune defense response in larvae of H. cunea by infectation, feeding and injection.
The P. pastoris expression system is currently the most successful eukaryotic expression system, capable of post-translational processing of the expressed proteins and enabling better protein activity than that obtained via prokaryotic expression [33]. To date, more than 500 exogenous proteins have been successfully expressed in this eukaryotic expression system [34,35]. The activity of the Pr1A protein expressed by Zhang et al. through P. pastoris was 10.6-fold higher than that of the Pr1A protein expressed in a prokaryotic expression system by Wang et al. [34,36]. In the present study, active CJPRB protein was obtained by expression in P. pastoris and could be used in subsequent experiments.
The PPO system plays a key role in insect immune defense by recognizing foreign invading substances. In addition, the protective enzymes SOD, POD and CAT are present in a dynamic balance in insects, and this dynamic balance plays an important role in maintaining the metabolic balance in insects. However, when insects are under stress, the protective enzyme system in the body changes or even disrupts the normal dynamic balance, leading to metabolic disorders in insects. In CJPRB-treated larvae of H. cunea, POD was the first enzyme to respond to CJPRB treatment, followed by an increase in CAT and SOD enzyme activities, while the PPO enzyme activity associated with insect humoral immunity was significantly higher than the control within 12-48 h of CJPRB treatment. In contrast, after CJPRB feeding of H. cunea larvae, CAT was the first to initiate protective effects, and POD acted slightly later, while SOD acted in the early and late stages of treatment. The PPO enzyme activity was significantly higher than that in the control group throughout the treatment and continued to act during the observation period after feeding. After CJPRB injection of H. cunea larvae, CAT, SOD and PPO responded to CJPRB treatment at an early stage and continued to respond for almost the entire observed post-treatment period, while POD showed a significant increase in activity only at 12 h and 36 h post-injection. In conclusion, this study showed that the order in which the protective enzyme systems responded to CJPRB treatment of H. cunea larvae differed greatly. However, the PPO enzyme activity was significantly higher than that in the control group in the differently treated H. cunea larvae for almost the whole observation period, which also indicated that CJPRB was able to elicit an immune defense response in the insects and to sustain its action. The study by St. Leger and Gillespie also showed that the Pr1 protease activates PPO in insects and oxidizes phenols to quinones and that sustained oxidation leads to insect mortality [37,38].
In general, the clearance of invasive pathogens by host insects requires four steps: recognition of the foreign pathogen, initiation of an extracellular reaction cascade that activates serine proteases and disarms serine protease inhibitors, activation of downstream Toll or Imd signaling pathways and expression of different AMPs [39]. In the process of resistance to CJPRB protease treatment, the initial upregulated expression of serine protease inhibitor (SP1-2) and serine protease (p1) in H. cunea, followed by initiation of signal transduction via pathways inducing upregulation of the expression of the AMP CEA and immune-related proteins, is consistent with the whole process of insect defense against foreign invasion. However, SP1-2 was upregulated at both 6 and 48 h. This was, presumably, because CJPRB is a serine protease, so the upregulated expression of SP1-2 at 6 h may have been due to the recognition of the CJPRB protein; after 48 h, SP1-2 was upregulated again, to prevent the inhibition of serine proteases in H. cunea due to insect autoimmune overload.
The midgut is an important physical barrier against pathogenic infections in insects, and local production of AMPs is one of the main defense mechanisms in the insect midgut [40]. The expression of the AMP CEA in H. cunea was consistently upregulated during 12-48 h of feeding, because CJPRB induced the expression of AMPs in H. cunea midgut cells. The expression of the immune-related protein HDD in H. cunea was upregulated during 12-60 h of feeding. In addition, chitin deacetylase, which was not upregulated in the infectation experiment, was upregulated after 12 and 60 h of feeding, showing that CJPRB feeding can induce CDA-2 (CDA1) and CDA-3 (CDA2) expression in the intestine of H. cunea. It has been shown that CDA-2 (CDA1) and CDA-3 (CDA2) are mainly expressed in the brown moth epidermis, which is different from our results [27]. In addition, the expression of the serine protease P1 was not high during feeding. However, SP1-2 showed upregulated expression at all time points except at 48 h and 72 h, showing that CJPRB may activate the immune effect of other serine proteases in insects during the feeding treatment.
The expression levels of most immune-related genes after CJPRB protein injection were higher than those of immune-related genes after both the infectation and feeding treatments. Among them, the H. cunea immune-related protein HDD was upregulated and consistently expressed throughout the observed phase, which is similar to the pattern of expression of this class of genes in H. cunea observed after E. coli injection [32]. In addition to HDD, CEA, CDA, P1 and SP1-2 were also upregulated during most of the observation period after CJPRB injection, a result that also indicates that blood cavity injection of CJPRB can trigger a more rapid and widespread immune response in insects than other methods.
In conclusion, this study showed that the subtilisin-like serine protease CJPRB from C. javanica BE01 was able to induce an immune response in H. cunea by infectation, feeding and injection. However, the mechanism by which CJPRB induced the immune response through interactions with H. cunea is still unclear, and we will follow up with further studies on the interactions of CJPRB proteins in H. cunea to explore the contribution of CJPRB to the infectation of H. cunea by C. javanica BE01.

Strains, Plasmids and Insects
The Pichia pastoris KM71H and E. coli Top10 strains were stored in the Pathology Laboratory of Nanjing Forestry University.
The P. pastoris expression vectors pPICZαA and pPICZαA-GFP and the expression vector PYF11-CJPRB [9] (CJPRB GenBank number: OM468894) were stored in the Pathology Laboratory of Nanjing Forestry University.
Healthy 2nd-and 5th-instar larvae of H. cunea (the eggs were provided by the Chinese Academy of Forestry Sciences; after hatching, the larvae were incubated under 25 ± 2 • C and 65 ± 5% RH with a 12:12 h L:D photoperiod) reared in captivity were used for the CJPRB bioassay experiments.

Construction of the Expression Vector pPICZαA-CJPRB and Transformation of the P. pastoris Strain KM71H
The PYF11-CJPRB plasmid was used as a template to amplify the full-length coding sequence (CDS) of CJPRB (OM468894) using the primers CJPRBF (CCGGAATTCCG-GATGCGCCTGTCCATCATCGCAGCAG) and CJPRBR (GCTCTAGAGCTCAATGATGAT-GATGATGATGATGATGAGTGGCGCCGTTGAAGGCCAGGTAG). The endonucleases EcoRI and Xbal were used to enzymatically cleave and linearize the vector pPICZαA. The pPICZαA vector was linearized by EcoRI and Xbal endonuclease. The CJPRB product obtained by PCR amplification and the linearized pPICZαA vector were recovered by using the EasyPure ® Quick Gel Extraction Kit (TransGen Biotech, Beijing, China). The recovered vector and CJPRB product were ligated overnight at 16 • C by using T4 ligase. The ligated product was transformed into competent E. coli Top10 cells, which were then coated on an LB selection plate containing kanamycin (50 µg/mL) and incubated overnight at 37 • C, after which the positive transformants were picked.
The pPICZαA-CJPRB plasmid was extracted from the positive transformants using the TIANprep Mini Plasmid Kit (TIANGEN, Beijing, China). Two micrograms of linearized pPICZαA-CJPRB (linearized by digestion with the endonuclease PmeI) was mixed with competent KM71H cells, and the mixture was transferred to a precooled 2 cm electrotransformation cup in an ice bath for 5 min. Electroconversion was performed using the following conditions: 1.5 kV voltage, 25 µV capacitance and 200 Ω resistance for 10 s. Immediately after electroconversion, 500 µL of prechilled 1 M sorbitol was added to the electrotransformation cup and mixed gently. The mixture was transferred to a sterilized centrifuge tube and shaken at 30 • C and 220 rpm for 1 h. The tube was centrifuged at 845× g for 3 min, and the cells were resuspended in a volume of 100 µL and spread uniformly on a YPD plate (containing 50 µg/mL bleomycin). The plate was incubated at 30 • C until a positive clone was produced.

Expression and Purification of the CJPRB Protein and GFP
A positive transformant (pPICZαA-CJPRB) was picked, added to 6 mL of liquid YPD medium (containing 50 µg/mL bleomycin) and incubated at 28 • C for 24 h with shaking at 200 rpm. One milliliter of the above-mentioned broth was inoculated into 240 mL of sterile BMGY medium in conical flasks (containing 30 mL of 1 M YNB); a total of 5 flasks were inoculated. After the culture was incubated for 24 h at 200 rpm and 28 • C, the supernatant was discarded, 120 mL of BMMY medium (containing 15 mL of 1 M YNB) was added to each of the 5 flasks and 1.2 mL of methanol was added to each flask to induce expression. Then 1.2 mL of methanol was added to each conical flask at regular intervals for 7 d at 200 rpm and 28 • C. On the 3rd, 4th, 5th, 6th and 7th days of methanol induction, one flask of bacterial culture was removed, and the supernatant was collected after centrifugation at 5432× g for 10 min. The centrifugation was repeated twice, and the final supernatant was collected.
GFP expression was induced as described above but only in one flask. Induction was performed by adding 1.2 mL of methanol per day, and the supernatant was collected after 3 d of induction (the induction conditions for GFP expression were determined in a previous study conducted in this laboratory).
CJPRB and GFPs were purified by Ni-NTA agarose purification resin preloaded onto gravity columns (Sheng Gong, Nanjing, China). The purified proteins (OD 280 > 1) were placed in dialysis bags and left overnight at 4 • C in 1 M PB buffer. Protein concentration assays were performed using the Pierce™ BCA Protein Assay Kit (Thermo, Waltham, MA, USA).

SDS-PAGE and Western Blot Analysis
The purified proteins (4 µL) were visualized by 10% and 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Thomas Brilliant Blue staining. For Western blot analysis, the recombinant CJPRB and GFPs were transferred to a polyvinylidene fluoride (PVDF) membrane at 200 mA for 2 h. The PVDF membrane was placed in 30 mL of blocking solution (1.2 g of skim milk powder dissolved in 1× PBST) and blocked at 4 • C overnight. The PVDF membrane was then removed, placed into 30 mL of blocking solution (1:5000) containing His-Tag (2A8) antibody (Abmart, Shanghai, China) and incubated on a lowspeed shaker for 1.5 h. The PVDF membrane was removed, placed into 1× PBST, and washed for 10 min on a low-speed shaker; this step was repeated three times. The PVDF membrane was then placed in 30 mL of blocking solution containing goat anti-mouse IgG (1:10,000; Abmart, Shanghai, China) and incubated on a low-speed shaker for 0.5 h. The membrane was then placed in 1× PBST and washed on a low-speed shaker for 10 min; this step was repeated three times. Finally, the PVDF membrane was scanned and imaged with a dual-channel infrared fluorescence scanning imager.

CJPRB Protein Activity Assay
The activity of the subtilisin-like serine protease CJPRB was assayed by the Folin phenol method [41]. The tyrosine standard curve was obtained according to Zhuang's method [41], with the equation y = 0.0047X + 0.00733, R 2 = 0.9955. The OD 680 value after the color development reaction was substituted into the standard curve equation to determine the concentration of tyrosine. Finally, the enzymatic activity of CJPRB was determined based on the tyrosine concentration. One unit of protease activity, expressed in U/mL, was defined as the amount of enzyme that produced 1 µg of tyrosine from the hydrolysis of casein in 1 min at 37 • C and pH 7.2.

CJPRB and GFP Infiltration of H. cunea Larvae
The 2nd-instar larvae of H. cunea were immersed in CJPRB (1 mg/mL) and GFP (1 mg/mL) solutions for 15 s, removed and placed on filter paper. Then the H. cunea larvae were placed into a rearing box after they had crawled and dried, and 0.1 g (approximately 30 heads) was sampled after 6 h, 12 h, 24 h, 36 h and 48 h.

CJPRB and GFP Feeding for H. cunea Larvae
The 2 × 2 × 2 cm square feed blocks were submerged in CJPRB (1 mg/mL) and GFP (1 mg/mL) protein solutions for 1 min and then removed after the feed had fully adsorbed the protein. The CJPRB-and GFP-treated feed was put into a feeder box and fed to starved H. cunea 2nd-instar larvae, and 0.1 g (approximately 30 heads) was sampled after 12 h, 24 h, 36 h, 48 h, 60 h and 72 h.

CJPRB and GFP Injection into H. cunea Larvae
Fifth-instar larvae of H. cunea (at the ventral segment of the third gastropod) were injected with 5 µL of protein per larva using a microsampler. Forty larvae were injected with CJPRB (1 mg/mL) and GFP (1 mg/mL) each, and 0.1 g was sampled after 6 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h.

Activity Assay of Protective Enzymes in H. cunea Larvae after CJPRB Treatment
The Tissue and Cell Protein Extraction Kit (Epizyme, Shanghai, China) was used to extract total protein from CJPRB-and GFP-treated larvae of H. cunea. The concentrations of the extracted proteins were determined using the BCA Protein Content Assay Kit (Ke ming, Suzhou, China).
SOD, POD, CAT and PPO assay kits (Kemin, Suzhou, China) were used to determine the activities of these enzymes in H. cunea after CJPRB treatment. GFP-treated H. cunea was used as a control.

RT-qPCR Validation of the Expression of Defense-Related Genes in H. cunea after CJPRB Protein Treatment
RNA extraction from larvae of H. cunea and cDNA synthesis were performed according to Wang's method [9]. The RT-qPCR system and conditions were as described by Wang [9]. The primers for the defense-related genes are shown in Table 1. The actin (Hcactin) gene of H. cunea was amplified as an internal control. The data were analyzed using the comparative threshold cycle (2−∆∆Ct) method. GFP-treated H. cunea was used as a control. Table 1. RT-qPCR primers used to verify the expression of defense-related genes of H. cunea.

Gene (GenBank Number)
F R